α-(1,3-dicarbonylenol ether) methyl ketones as cysteine protease inhibitors

ABSTRACT

Cysteine protease inhibitors which deactivate the protease by covalently bonding to the cysteine protease and releasing the enolate of a 1,3-dicarbonyl (or its enolic form). The cysteine protease inhibitors of the present invention accordingly comprise a first portion which targets a desired cysteine protease and positions the inhibitor near the thiolate anion portion of the active site of the protease, and a second portion which covalently bonds to the cysteine protease and irreversibly deactivates that protease by providing a carbonyl or carbonyl-equivalent which is attacked by the thiolate anion of the active site of the cysteine protease to sequentially cleave a β-dicarbonyl enol ether leaving group.

RELATION TO PENDING APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/164,031, filed Dec. 8, 1993, now U.S. Pat. No. 5,486,623.

FIELD OF THE INVENTION

The present invention relates generally to cysteine protease inhibitors,and more particularly to cysteine protease inhibitors which are peptidylketones which contain dicarbonyl enolether leaving groups. The cysteineprotease inhibitors of the present invention are particularly designedfor the in vivo management of cysteine proteases, particularlycathepsins B, L, H and C, calpains I and II, interkeukin 1-β-convetingenzyme ("ICE"), and the primitive enzymatic counterparts of thesecysteine proteases.

BACKGROUND TO THE INVENTION

Cysteine proteases associated with human disease states can be groupedinto three categories: (1) lysosomal cathepsins; (2) cytosolic calpainsand processing enzymes such as interkeukin conveting enzymes; and (3)prokaryotic enzymes with autocatalytic activation. Cathepsins B, H, andL are cysteinyl proteases involved in normal protein degradation. Assuch, they are generally located in the lysosomes of cells. When theseenzymes are found extralysosomaly they have been implicated by use ofsynthetic substrate technology and by natural endogenous inhibitors asplaying a causative role in a number of disease states such asrheumatoid arthritis, osteo arthritis, pneumocystis carinii,schistosomiasis, trypanosoma cruzi, trypanosoma brucei brucei, Crithidiafusiculata, malaria, periodontal disease, tumor metastasis,metachromatic leukodystrophy, muscular dystrophy, etc. For example, aconnection between cathepsin B-type enzymes and rheumatoid arthritis hasbeen suggested in van Noorden and Everts, "Selective Inhibition ofCysteine Proteinases by Z-Phe-Ala-CH₂ F Suppresses Digestion of Collagenby Fibroblasts and Osteoclasts," 178 Biochemical and BiophysicalResearch Communications 178; Rifkin, Vernillo, Kleekner, Auszmann,Rosenberg and Zimmerman, "Cathepsin B and L Activities in IsolatedOsteoclasts," 179 Biochemical and Biophysical Research Communications63; Grinde, "The Thiol Proteinase Inhibitors, Z-Phe-Phe-CHN₂ andZ-Phe-Ala-CHN₂, Inhibit Lysosomal Protein Degradation in Isolated RatHepatocytes," 757 Biochimica et Biophysica Acta 15; Mason, Bartholomewand Hardwick, "The Use of Benzyloxycarbonyl ¹²⁵I!iodotyrosylalanyldiazomethane as a Probe for Active CysteineProteinases in Human Tissues," 263 Biochem. J. 945; van Noorden, Smithand Rasnick, "Cysteine Proteinase Activity in Arthritic Rat Knee Jointsand the Effects of a Selective Systemic Inhibitor, Z-Phe-Ala-CH₂ F," 15J. Rheumatol. 1525; and van Noorden, Vogels and Smith, "Localization andCytophotometric Analysis of Cathepsin B Activity in Unfixed andUndecalified Cryostat Sections of Whole Rat Knee Joints," 37 J.Histochemistry and Cytochemistry 617. A connection between cathepsin Band osteo arthritis has been suggested in Delaisse, Eeckhout and Vaes,"In Vivo and In Vitro Evidence for the Involvement of CysteineProteinases in Bone Resorption," 125 Biochemical and BiophysicalResearch Communications 441; a connection between cathepsin B andpneumocystis carinii has been suggested in Hayes, Stubberfield, McBrideand Wilson, "Alterations in Cysteine Proteinase Content of Rat LungAssociated with Development of Pneumocystis Carinii Infection," 59Infection and Immunity 3581; a connection between cysteine proteinasesand schistosomiasis has been suggested in Cohen, Gregoret, Amiri,Aldape, Railey and McKerrow, "Arresting Tissue Invasion of a Parasite byProtease Inhibitors Chosen With the Aid of Computer Modeling," 30Biochemistry 11221. A connection between cysteine proteinases andtrypanosoma cruzi, trypanosoma brucei brucei and crithidia fasciculatahas been suggested in Ashall, Harris, Roberts, Healy and Shaw,"Substrate Specificity and Inhibitor Sensitivity of a TrypanosomatidAlkaline Peptidase," 1035 Biochimica et Biphysica Acta 293, and/or inAshall, Angliker and Shaw, "Lysis of Trypanosomes by PeptidylFluoromethyl Ketones," 170 Biochemical and Biophysical ResearchCommunications 923. A connection between cysteine proteinases andmalaria has been suggested in Rosenthal, Wollish, Palmer and Rasnick,"Antimalarial Effects of Peptide Inhibitors of a Plasmodium FalciparumCysteine Proteinase," 88 J. Clin. Invest. 1467, and in Rosenthal, Leeand Smith, "Inhibition of a Plasmodium Vinckei Cysteine Proteinase CuresMurine Malaria," 91 J. Clin. Invest. 1052. A connection betweencathepsin B and tumor metathesis has been suggested in Smith, Rasnick,Burdick, Cho, Rose and Vahratian, "Visualization of Time-DependentInactivation of Human Tumor Cathepsin B Isozymes by a PeptidylFluoromethyl Ketone Using a Fluorescent Print Technique," 8 Anti-cancerResearch 525. A connection between cathepsin B and cancer has beensuggested in Gordon and Mourad, 2 Blood Coagulation and Fibrinolysis735. A connection between cathepsin B and periodontal disease has beensuggested in Cox, Cho, Eley and Smith, "A Simple, Combined Fluorogenicand Chromogenic Method for the Assay of Proteases in Gingival CrevicularFluid," 25 J. Periodont. Res. 164; Uitto, Larjava, Heino and Sorsa, "AProtease of Bacteroides Gingivalis Degrades Cell Surface and MatrixGlycoproteins of Cultured Gingival Fibroblasts and Induces Secretion ofCollagenase and Plasminogen Activator," 57 Infection and Immunity 213;Kunimatsu, Yamamoto, Ichimaru, Karo and Karo, "Cathepsins B, H and LActivities in Gingival Crevicular Fluid From Chronic Adult PeriodontitisPatients and Experimental Gingivitis Subjects," 25 J Peridont Res 69;Beighton, Radford and Naylor, "Protease Activity in Gingival CrevicularFluid From Discrete Periodontal Sites in Humans With Periodontitis orGingivitis"; 35 Archs oral Biol. 329; Cox and Eley, "Preliminary Studieson Cysteine and Serine Proteinase Activities in Inflamed Human GingivaUsing Different 7-Amino-4-Trifluoromethyl Coumarin Substrates andProtease Inhibitors," 32 Archs oral Biol. 599; and Eisenhauer,Hutchinson, Javed and McDonald, "Identification of a Cathepsin B-LikeProtease in the Crevicular Fluid of Gingivitis Patients," 62 J Dent Res917. A connection between cathepsin B and metachromatic leukodystrophyhas been suggested in von Figura, Steckel, Conary, Hasilik and Shaw,"Heterogeneity in Late-Onset Metachromatic Leukodystrophy. Effect ofInhibitors of Cysteine Proteinases," 39 Am J Hum Genet. 371; aconnection between cathepsin B and muscular leukodystrophy has beensuggested in Valentine, Winand, Pradhan, Moise, de Lahunta, Kornegay andCooper, "Canine X-Linked Muscular Dystrophy as an Animal Model ofDuchenne Muscular Dystrophy: A Review," 42 Am J Hum Genet 352; aconnection between cathepsin B and rhinovirus has been suggested inKnott, Orr, Montgomery, Sullivan and Weston, "The Expression andPurification of Human Rhinovirus Protease 3C," 182 Eur. J. Biochem. 547;a connection between cathepsin B and kidney disease has been suggestedin Baricos, O'Connor, Cortez, Wu and Shah, "The Cysteine ProteinaseInhibitor, E-64, Reduces Proteinuria in an Experimental Model ofGlomerulonephritis," 155 Biochemical and Biophysical ResearchCommunications 1318; and a connection between cathepsin B and multiplesclerosis has been suggested in Dahlman, Rutschmann, Kuehn and Reinauer,"Activation of the Multicatalytic Proteinase from Rat Skeletal Muscle byFatty Acids or Sodium Dodecyl Sulphate," 228 Biochem. J. 171.

Connections between certain disease states and cathepsins H and C havealso been established. For example, cathepsin H has been directly linkedto the causative agents of Pneumocystis carinii and in the neuromusculardiseases Duchenne dystrophy, polymyositis, and neurogenic disorders.Stauber, Riggs and Schochet, "Fluorescent Protease Histochemistry inNeuromuscular Disease," Neurology 194 (Suppl. 1) March 1984; Stauber,Schochet, Riggs, Gutmann and Crosby, "Nemaline Rod Myopathy: Evidencefor a Protease Deficiency," Neurology 34 (Suppl. 1) March 1984.Similarly, cathepsin C has been directly linked to muscular diseasessuch as nemaline myopathy, to viral infections, and to processing andactivation of bone marrow serine proteases (elastase and granzyme A).McGuire, Lipsky and Thiele, "Generation of Active Myeloid and LymphodGranule Serine Proteases Requires Processing by the Granule ThiolProtease Dipeptidyl Peptidase I, 268 J. Biol. Chem. 2458-67; L. Polgar,Mechanisms of Protease Action (1989); Brown, McGuire and Thiele,"Dipeptidyl Peptidase I is Enriched in Granules of In Vitro- and InVivo-Activated Cytotoxic T Lymphocytes," 150 Immunology 4733-42. TheBrown et al. study effectively demonstrated the feasibility ofinhibiting cathepsin C (DPP-I) in the presence of other cysteinylenzymes based on substrate specificity. Unfortunately, the diazoketonesused in that study are believed to be mutagenic and not appropriate forin vivo application.

The cytosolic or membrane-bound cysteine proteases called calpains havealso been implicated in a number of disease states. For example, calpaininhibitor can be useful for the treatment of muscular disease such asmuscular dystrophy, amyotrophy or the like, 25 Taisha (Metabolism) 183(1988); 10 J. Pharm. Dynamics 678 (1987); for the treatment of ischemicdiseases such as cardiac infarction, stroke and the like, 312 New Eng.J. Med. 159 (1985); 43 Salshin Igaku 783 (1988); 36 ArzneimittelForschung/Drug Research 190, 671 (1986); 526 Brain Research 177 (1990);for improving the consciousness disturbance or motor disturbance causedby brain trauma, 16 Neurochemical Research 483 (1991); 65 J.Neurosurgery 92 (1986); for the treatment of diseases caused by thedemyelination of neurocytes such as multiple sclerosis, peripheralnervous neuropathy and the like, 47 J. Neurochemistry 1007 (1986); andfor the treatment of cataracts, 28 Investigative Ophthalmology & VisualScience 1702 (1987); 34 Experimental Eye Research 413 (1982); 6 Lens andEye Toxicity Research 725 (1989): 32 Investigative Ophthalmology &Visual Science 533 (1991).

Calpain inhibitors may also be used as therapeutic agents for fulminanthepatitis, as inhibitors against aggregation of platelet caused bythrombin, 57 Thrombosis Research 847 (1990); and as a therapeutic agentfor diseases such as breast carcinoma, prostatic carcinoma orprostatomegaly, which are suspected of being caused by an abnormalactivation of the sex hormone receptors.

Certain protease inhibitors have also been associated with Alzheimer'sdisease. See, e.g., 11 Scientific American 40 (1991). Further, thiolprotease inhibitors are believed to be useful as anti-inflammatorydrugs, 263 J. Biological Chem. 1915 (1988); 98 J. Biochem. 87 (1985); asantiallergic drugs, 42 J. Antibiotics 1362 (1989); and to prevent themetastasis of cancer, 57 Seikagaku 1202 (1985); Tumor Progression andMarkers 47 (1982); and 256 J. Biological Chemistry 8536 (1984).

Further, Interleukin 1-β-Converting Enzyme (ICE) has been shown to be acysteine protease implicated in the formation of the cytokine IL-1βwhich is a potent mediator in the pathogenesis of chronic and acuteinflammatory diseases. Tocci and Schmidt, ICOP Newsletter, September1994. Inhibitors to this enzyme have recently been reported, includingThornberry, Peterson, Zhao, Howard, Griffin, and Chapman, "Inactivationof Interleukin-1β-Converting Enzyme by Peptide (Acyloxy)methyl Ketones,33 Biochemistry 3934 (1994); Dolle, Singh, Rinker, Hoyer, Prasad,Graybill, Salvino, Helaszek, Miller and Ator, "Aspartylα-((1-Phenyl-3-(trifluoromethyl)-pyrazol-5-yl)-oxy)methyl Ketones asInterleukin-1β Converting Enzyme Inhibitors: Significance of the P₁ andP₃ Amido Nitrogens for Enzyme-Peptide Inhibitor Binding" 37 J. Med.Chem. 3863; Mjalli, Chapman, MacCoss, Thornberry, Peterson, "ActivatedKetones as Potent Reversible Inhibitors of Interleukin-1β-ConvertingEnzyme" 4 Biooganic & Medicinal Chemistry Letters, 1965; and Dolle,Singh, Whipple, Osifo, Speier, Graybill, Gregory, Harris, Helaszek,Miller and Ator "Aspartyl α-((Diphenylphosphinyl)-oxy)-methyl Ketones asNovel Inhibitors of Interleukin-1β-Converting Enzyme: Utility of theDiphenylphosphionic Acid Leaving Group for the Inhibition of CysteineProteases" 38 J. Med. Chem. 220.

The most promising type of cysteine proteinase inhibitors have anactivated carbonyl with a suitable α-leaving group fused to a programmedpeptide sequence that specifically directs the inhibitor to the activesite of the targeted enzyme. Once inside the active site, the inhibitorcarbonyl is attacked by a cysteine thiolate anion to give the resultinghemiacetal, which collapses via a 1,2-thermal migration of the thiolateand subsequent displacement of the α-keto-leaving group. The bondbetween enzyme and inhibitor is then permanent and the enzyme isirreversibly inactivated.

The usefulness of an inhibitor in inactivating a particular enzymetherefore depends not only on the "lock and key" fit of the peptideportion, but also on the reactivity of the bond holding the α-leavinggroup to the rest of the inhibitor. It is important that the leavinggroup be reactive only to the intramolecular displacement via a1,2-migration of sulfur in the breakdown of the hemithioacetalintermediate.

Groundbreaking work regarding cysteine proteinase inhibitors having anactivated carbonyl, a suitable α-leaving group and a peptide sequencethat effectively and specifically directs the inhibitor to the activesite of the targeted enzyme was disclosed in U.S. Pat. No. 4,518,528 toRasnick, incorporated herein by reference. That patent establishedpeptidyl fluoromethyl ketones to be unprecedented inhibitors of cysteineproteinase in selectivity and effectiveness. The fluoromethyl ketonesdescribed and synthesized by Rasnick included those of the formula:##STR1## wherein R₁ and R₂ are independently selected from the grouphydrogen, alkyl of 1-6 carbons, substituted alkyl of 1-6 carbons, aryl,and alkylaryl where the alkyl group is of 1-4 carbons; n is an integerfrom 1-4 inclusive; X is a peptide end-blocking group; and Y is an aminoacid or peptide chain of from 1-6 amino acids.

Peptidylketone inhibitors using a phenol leaving group are similar tothe peptidyl fluoroketones. As is known in the art, oxygen most closelyapproaches fluorine in size and electronegativity. Further, when oxygenis bonded to an aromatic ring these values of electronegativity becomeeven closer due to the electron withdrawing effect of the sp² carbons.The inductive effect of an α-ketophenol versus an α-ketofluoride whenmeasured by the pKa of the α-hydrogen, appears comparable withinexperimental error.

Unfortunately, the leaving groups of prior art inhibitors that use aphenoxy group present problems of toxicity, solubility, etc. Solubilityis of particular importance in the field of peptide derived drugs wherebioavailability becomes the major criterion for the success of a drug.The solubility recommendation of the FDA is 5 mg/mL. Successful in vivoutility of prior art inhibitors has been limited due to the insolubilityof the leaving groups. In vivo application to date has centered oninhibitors with peptide requirements allowing ester, acid or free amineside chains as those required in the inhibition ofInterleukin-1β-converting enzyme: Revesz, Briswalter, Heng, Leutwiler,Mueller and Wuethrich, "35 Tetrahedron Letters 9693.

International application WO 93/09135 disclosed inhibitors againdesigned for Interleukin-1β-converting enzyme where anN-hydroxytetrazole was disclosed as a leaving group. Further, tetrazoleshave also been used in other pharmaceutical products such as Ceforanide,etc.

The in vivo inhibition of other cysteine proteases using oxygen anionicleaving groups was first disclosed by Zimmerman, Bissell, and Smith inU.S. Pat. No. 5,374,623 where it was disclosed that bioavailability isenhanced by the use of peptidyl α-aromatic ether methyl ketones withselective peptide combinations not requiring the presence of a freeamine or acid side chain. Later, a peptidyl (acyloxy)methyl ketone withlysine in the side chain was reported to have in vivo efficacy: Wagner,Smith, Coles, Copp, Ernest and Krantz, "In Vivo Inhibition of CathepsinB by Peptidyl (Acyloxy)methyl Ketones," 37 J. Med. Chem. 1833.Unfortunately, peptidyl (acyloxy)methyl ketones are esters that are alsosubject to cleavage by esterases which makes the α-ketoethers thepreferred construction for cysteine protease inhibitors.

It can be seen from the foregoing that a need continues to exist forcysteine protease inhibitors with improved solubility and toxicityprofiles, and which are particularly suitable for in vivo use. Thepresent invention addresses that need.

SUMMARY OF THE INVENTION

Briefly describing the present invention, there is provided a class ofcysteine protease inhibitors which deactivates the protease bycovalently bonding to the cysteine protease and releasing the enolate ofa 1,3-dicarbonyl (or its enolic form). The cysteine protease inhibitorsof the present invention accordingly comprise a first portion whichtargets a desired cysteine protease and positions the inhibitor near thethiolate anion portion of the active site of the protease, and a secondportion which covalently bonds to the cysteine protease and irreversiblydeactivates that protease by providing a carbonyl or carbonyl-equivalentwhich is attacked by the thiolate anion of the active site of thecysteine protease to sequentially cleave a β-dicarbonyl enol etherleaving group.

The cysteine protease inhibitors of the present invention may be definedby the formula below: ##STR2## where B is H or an N-terminal blockinggroup;

R₁₋₃ are the amino acid side chains of the P1-3 amino acids,respectively;

n is 0 or 1;

m is 0 or 1; and

G is a five- or six-membered ring portion of the β-dicarbonyl enol etherleaving group as defined by the formulas below.

In one embodiment, the compositions of the present invention arecathepsin or calpain inhibitors of the formula: ##STR3## where B is H oran N-terminal blocking group;

R₁ is the amino acid side chain of the P₁ amino acid residue; whereinthe P₁ amino acid is not Asp;

each P_(n) is an amino acid residue, or is a heterocyclic replacement ofthe amino acid wherein the heterocycle is a piperazine, adecahydroisoquinoline, a pyrrolinone, a pyridine, a pyridone, acarbolinone, a quinazoline, a pyrimidone or the like;

m is 0 or a positive integer;

R₄ is a hydroxyl, alkoxyl, acyl, hydrogen, alkyl or phenyl;

R₅ and R₆ are jointly a carboxyl group or a double bond terminating inan alkyl or an aryl group, or are independently acyl, aryl or heteroarylif R₄ is hydrogen, alkyl or phenyl, or are independently acyl, alkyl,hydrogen, aryl or heteroaryl otherwise; and

X is N, S, O or CH₂.

In another embodiment, the compositions of the present invention are ICEinhibitors of the formula: ##STR4## where B is H or an N-terminalblocking group;

R₁ is the Asp amino acid side chain;

each P_(n) is an amino acid residue, or is a heterocyclic replacement ofthe amino acid wherein the heterocycle is a piperazine, adecahydroisoquinoline, a pyrrolinone, a pyridine, a pyridone, acarbolinone, a quinazoline, a pyrimidone or the like;

m is 0 or a positive integer;

R₄ is a hydroxyl, alkoxyl, acyl, hydrogen, alkyl or phenyl;

R₅ and R₆ are jointly a carboxyl group or a double bond terminating inan alkyl or an aryl group, or are independently acyl, aryl or heteroarylif R₄ is hydrogen, alkyl or phenyl, or are independently acyl, alkyl,hydrogen, aryl or heteroaryl otherwise; and

X is N, S, O or CH₂.

In another embodiment, the cysteine protease inhibitor is of theformula: ##STR5## where B is H or an N-terminal blocking group;

each P_(n) is an amino acid residue, or is a heterocyclic replacement ofthe amino acid wherein the heterocycle is a piperazine, adecahydroisoquinoline, a pyrrolinone, a pyridine, a pyridone, acarbolinone, a quinazoline, a pyrimidone or the like;

m is 0 or a positive integer;

R₄ is a hydroxyl, alkoxyl, acyl, hydrogen, alkyl or phenyl;

R₁₀ is H or an optionally substituted alkyl, aryl, heteroaryl, or theresidue of a sugar; and

X is N, S, O or CH₂.

In another embodiment, the cysteine protease inhibitor is of theformula: ##STR6## where B is H or an N-terminal blocking group;

each P_(n) is an amino acid residue, or is a heterocyclic replacement ofthe amino acid wherein the heterocycle is a piperazine, adecahydroisoquinoline, a pyrrolinone, a pyridine, a pyridone, acarbolinone, a quinazoline, a pyrimidone or the like;

m is 0 or a positive integer;

R₅ and R₆ are independently hydrogen, alkyl or acyl; and

X is N, S, O or CH₂.

In another embodiment, the cysteine protease inhibitor is of theformula: ##STR7## where B is H or an N-terminal blocking group;

each P_(n) is an amino acid residue, or is a heterocyclic replacement ofthe amino acid wherein the heterocycle is a piperazine, adecahydroisoquinoline, a pyrrolinone, a pyridine, a pyridone, acarbolinone, a quinazoline, a pyrimidone or the like;

m is 0 or a positive integer;

R₂ and R₃ are indepentantly H or an alkyl or alkenyl group; and

X is N, S, O or CH₂.

In another embodiment, the cysteine protease inhibitors are of theformula: ##STR8## where B is H or an N-terminal blocking group;

each P_(n) is an amino acid residue, or is a heterocyclic replacement ofthe amino acid wherein the heterocycle is a piperazine, adecahydroisoquinoline, a pyrrolinone, a pyridine, a pyridone, acarbolinone, a quinazoline, a pyrimidone or the like;

m is 0 or a positive integer;

R₄ is a hydroxyl, alkoxyl, acyl, hydrogen, alkyl or phenyl;

R₅, R₆ R₇ and R₈ are independently hydrogen, alkyl, acyl, phenyl, halo,hydroxyl, oxy or alkoxy; and

X is N, S, O or CH₂.

In another embodiment, the cysteine protease inhibitors are of theformula: ##STR9## where B is H or an N-terminal blocking group;

each P_(n) is an amino acid residue, or is a heterocyclic replacement ofthe amino acid wherein the heterocycle is a piperazine, adecahydroisoquinoline, a pyrrolinone, a pyridine, a pyridone, acarbolinone, a quinazoline, a pyrimidone or the like;

m is 0 or a positive integer;

R₄ is a hydroxyl, alkoxyl, acyl, hydrogen, alkyl or phenyl;

R₅ and R₆ may be attached to R₇ and R₈ to form a ring that is eithersaturated or unsaturated or aromatic; and

X is N, S, O or CH₂.

In another embodiment, the cysteine protease inhibitors are of theformula: ##STR10## where B is H or an N-terminal blocking group;

each P_(n) is an amino acid residue, or is a heterocyclic replacement ofthe amino acid wherein the heterocycle is a piperazine, adecahydroisoquinoline, a pyrrolinone, a pyridine, a pyridone, acarbolinone, a quinazoline, a pyrimidone or the like;

m is 0 or a positive integer;

R₄ is a hydroxyl, alkoxyl, acyl, hydrogen, alkyl or phenyl;

R₅ and R₈ are independently hydrogen, alkyl, acyl, phenyl, halo,hydroxyl, oxy or alkoxy, or R₅ is attached to R₈ to form a homocyclic orhererocyclic ring that is either saturated or unsaturated or aromatic;and

X is N, S, O or CH₂.

One object of the present invention is to provide improved cysteineprotease inhibitors with improved solubility and toxicity profiles.

A further object of the present invention is to provide a class ofcysteine protease inhibitors which are particularly effective for invivo applications.

Further objects and advantages of the present invention will be apparentfrom the following description.

DESCRIPTION OF THE PREFERRED EMBODIMENT

For the purposes of promoting an understanding of the principles of theinvention, reference will now be made to preferred embodiments andspecific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of theinvention is thereby intended, such alterations and furthermodifications of the invention, and such further applications of theprinciples of the invention as illustrated herein, being contemplated aswould normally occur to one skilled in the art to which the inventionrelates.

As indicated above, the present invention relates to cysteine proteaseinhibitors which contain 1,3-dicarbonyl enolether leaving groups. In oneaspect of the invention, a group of cysteine protease inhibitors whichhave been shown to be particularly effective for in vivo applications isdisclosed.

The cysteine protease inhibitors described herein function as the sum oftwo portions. The first portion defines the specificity of a particularinhibitor to an enzyme by the spacial, hydrophobic or hydrophilic andionic interactions of a particular composition that either imitates orimproves upon the nature of the enzyme's natural substrate. The secondportion is a trap that covalently binds the enzyme in a two-stepmechanism: the first step involves the nucleophilic attack of the enzymethiolate on the carbonyl of the inhibitor to form a hemithioketal. It isthen energetically favorable for this intermediate to undergo a 1,2migration of the thiolate in which an enolate (or enol form) of a1,3-dicarbonyl is released. The enzyme has now become irreversiblybonded to the inhibitor. With the inhibitors of the present inventionthe leaving group is the enol form of a 1,3-dicarbonyl.

Accordingly, the cysteine proteinase inhibitors of the present inventionare preferably constructed with an activated carbonyl which bears asuitable α-leaving group which is fused to a programmed peptide sequencethat specifically directs the inhibitor to the active site of thetargeted enzyme. (For example, Z-Phe-PheCHN₂ preferentially inhibitscathepsin L over cathepsin B.) Once inside the active site, thisinhibitor carbonyl is attacked by a cysteine thiolate anion to give theresulting hemiacetal form. If the α-leaving group then breaks off, thebond between enzyme and inhibitor becomes permanent and the enzyme isirreversibly inactivated. The selectivity of the inhibitor for aparticular enzyme depends not only on the "lock and key" fit of thepeptide portion, but also on the reactivity of the bond binding theleaving group to the rest of the inhibitor. It is very important thatthe leaving group must be reactive to the intramolecular displacementvia a 1,2-migration of sulfur in the breakdown of the hemithioacetalintermediate. The mechanism of protease inhibition is shown below inFIG. 1. ##STR11##

The preferred inhibitors of the present invention can be describedgenerally by the formula: ##STR12## where B is H or an N-terminalblocking group;

R₁₋₃ are the amino acid side chains of the P₁₋₃ amino acids,respectively;

n is 0 or 1;

m is 0 or 1; and

X is a five- or six-membered ring portion of the β-dicarbonyl enol etherleaving group, as further defined below.

In one embodiment, the compositions of the present invention arecathepsin or calpain inhibitors of the formula: ##STR13## where B is Hor an N-terminal blocking group;

R₁ is the amino acid side chain of the P₁ amino acid residue; whereinthe P₁ amino acid is not Asp; each P_(n) is an amino acid residue, or isa heterocyclic replacement of the amino acid wherein the heterocycle isa piperazine, a decahydroisoquinoline, a pyrrolinone, a pyridine, acarbolinone, a quinazoline, a pyrimidone or the like;

m is 0 or a positive integer;

R₄ is a hydroxyl, alkoxyl, acyl, hydrogen, alkyl or phenyl;

R₅ and R₆ are jointly a carboxyl group or a double bond terminating inan alkyl or an aryl group, or are independently acyl, aryl or heteroarylif R₄ is hydrogen, alkyl or phenyl, or are independently acyl, alkyl,hydrogen, aryl or heteroaryl otherwise; and

X is N, S, O or CH₂.

In another embodiment, the compositions of the present invention are ICEinhibitors of the formula: ##STR14## where B is H or an N-terminalblocking group;

R₁ is the Asp amino acid side chain;

each P_(n) is an amino acid residue, or is a heterocyclic replacement ofthe amino acid wherein the heterocycle is a piperazine, adecahydroisoquinoline, a pyrrolinone, a pyridine, a carbolinone, aquinazoline, a pyrimidone or the like;

m is 0 or a positive integer;

R₄ is a hydroxyl, alkoxyl, acyl, hydrogen, alkyl or phenyl;

R₅ and R₆ are jointly a carboxyl group or a double bond terminating inan alkyl or an aryl group, or are independently acyl, aryl or heteroarylif R₄ is hydrogen, alkyl or phenyl, or are independently acyl, alkyl,hydrogen, aryl or heteroaryl otherwise; and

X is N, S, O or CH₂.

In another embodiment, the cysteine protease inhibitor is of theformula: ##STR15## where B is H or an N-terminal blocking group;

each P_(n) is an amino acid residue, or is a heterocyclic replacement ofthe amino acid wherein the heterocycle is a piperazine, adecahydroisoquinoline, a pyrrolinone, a pyridine, a carbolinone, aquinazoline, a pyrimidone or the like;

m is 0 or a positive integer;

R₄ is a hydroxyl, alkoxyl, acyl, hydrogen, alkyl or phenyl;

R₁₀ is H or an optionally substituted alkyl, aryl, heteroaryl, or theresidue of a sugar; and

X is N, S, O or CH₂.

In another embodiment, the cysteine protease inhibitor is of theformula: ##STR16## where B is H or an N-terminal blocking group;

each P_(n) is an amino acid residue, or is a heterocyclic replacement ofthe amino acid wherein the heterocycle is a piperazine, adecahydroisoquinoline, a pyrrolinone, a pyridine, a carbolinone, aquinazoline, a pyrimidone or the like;

m is 0 or a positive integer;

R₅ and R₆ are independently hydrogen, alkyl or acyl; and

X is N, S, O or CH₂.

Most preferably, R₅ and R₆ are each hydrogen. In one alternativeembodiment the H on the hydroxyl group of the heterocyclic leaving groupmay be replaced by an alkyl or ankenyl group.

In another embodiment, the cysteine protease inhibitor is of theformula: ##STR17## where B is H or an N-terminal blocking group;

each P_(n) is an amino acid residue, or is a heterocyclic replacement ofthe amino acid wherein the heterocycle is a piperazine, adecahydroisoquinoline, a pyrrolinone, a pyridine, a carbolinone, aquinazoline, a pyrimidone or the like;

m is 0 or a positive integer;

R₂ and R₃ are indepentantly H or an alkyl or alkenyl group; and

X is N, S, O or CH₂.

Most preferably R₅ is CH₃ and R₆ is C₂ H₅ as shown in compound A2,infra.

In another embodiment, the cysteine protease inhibitors are of theformula: ##STR18## where B is H or an N-terminal blocking group;

each P_(n) is an amino acid residue, or is a heterocyclic replacement ofthe amino acid wherein the heterocycle is a piperazine, adecahydroisoquinoline, a pyrrolinone, a pyridine, a carbolinone, aquinazoline, a pyrimidone or the like;

m is 0 or a positive integer;

R₄ is a hydroxyl, alkoxyl, acyl, hydrogen, alkyl or phenyl;

R₅, R₆, R₇ and R₈ are independently hydrogen, alkyl, acyl, phenyl, halo,hydroxyl, oxy or alkoxy; and

X is N, S, O or CH₂.

In another embodiment, the cysteine protease inhibitors are of theformula: ##STR19## where B is H or an N-terminal blocking group;

each P_(n) is an amino acid residue, or is a heterocyclic replacement ofthe amino acid wherein the heterocycle is a piperazine, adecahydroisoquinoline, a pyrrolinone, a pyridine, a carbolinone, aquinazoline, a pyrimidone or the like;

m is 0 or a positive integer;

R₄ is a hydroxyl, alkoxyl, acyl, hydrogen, alkyl or phenyl;

R₅ and R₆ may be attached to R₇ and R₈ to form a ring that is eithersaturated or unsaturated or aromatic; and

X is N, S, O or CH₂.

In another embodiment, the cysteine protease inhibitors are of theformula: ##STR20## where B is H or an N-terminal blocking group;

each P_(n) is an amino acid residue, or is a heterocyclic replacement ofthe amino acid wherein the heterocycle is a piperazine, adecahydroisoquinoline, a pyrrolinone, a pyridine, a carbolinone, aquinazoline, a pyrimidone or the like;

m is 0 or a positive integer;

R₄ is a hydroxyl, alkoxyl, acyl, hydrogen, alkyl or phenyl;

R₅ and R₈ are independently hydrogen, alkyl, acyl, phenyl, halo,hydroxyl, oxy or alkoxy, or R₅ is attached to R₈ to form a homocyclic orhererocyclic ring that is either saturated or unsaturated or aromatic;and

X is N, S, O or CH₂.

As to the amino acid blocking group B for the N-terminal amino acidnitrogen, many suitable peptide end-blocking groups are known in theart. For example the end-blocking groups identified in E. Gross and J.Meienhofer (eds.), The Peptides, Vol. 3 are generally suitable for usein the present invention. Preferred blocking groups include N-morpholinecarbonyl and derivatives of propionic acid derivatives that haveintrinsic analgesic or anti-inflammatory action. Examples of blockinggroups having intrinsic analgesic or anti-inflammatory action may befound in Gilman, Goodman, Gilman, The Pharmacological Basis ofTherapeutics, Sixth Ed. MacMillan, Chapter 29. As defined herein, thepeptide end-blocking group is attached to either an amino acid or apeptide chain.

One particularly effective blocking group is the 4-morpholinylcarbonyl("Mu") blocking group shown below: ##STR21##

Other useful blocking groups include the morphine sulfonyl group andrelated groups as reported by Doherty et al. in "Design and Synthesis ofPotent, Selective and Orally Active Fluorine-Containing ReninInhibitors," 35 J. Med. Chem. 2. An appropriate blocking group for aparticular inhibitor may be selected by persons skilled in the artwithout undue experimentation.

As is conventional in the art, and as used herein, amino acid residuesare generally designated as P₁, P₂, etc., wherein P₁ refers to the aminoacid residue nearest the leaving group, P₂ refers to the amino acidresidue next to P₁ and nearer the blocking group, etc. In dipeptideinhibitors therefore, P₂ is the amino acid residue nearest the blockinggroup. In this disclosure the chain of amino acid residues is frequentlywritten (P_(n))_(m) with each P_(n) being an amino acid residue and mbeing zero or a positive integer. Each P_(n) may, of course, be adifferent amino acid residue. Preferably, m is less than or equal tofour. Most preferably, m is two.

As suggested above, any of the amino acid residues may be replaced by aheterocyclic replacement. Preferably the heterocycle is a piperazine, adecahydroisoquinoline, a pyrrolinone, a pyridine, a carbolinone, aquinazoline, a pyrimidone or the like. Persons skilled in the art mayselect an appropriate heterocycle in a manner similar to that in whichappropriate amino acid residues are selected. As used herein therefore,terminology such as "the peptide portion" refers as well to thecorresponding portion when heterocycles replace any or all of the aminoacids.

The peptide portion of the inhibitor includes any peptide appropriatefor targetting a desired cysteine protease. In particular, the sidechain on the P₁ amino acid is selected according to the enzyme beingtargetted. For cathepsin B or L, this might include side chains suchthat the linked P₁ amino acid is a member of the group consisting ofalanyl (Ala), arginyl (Arg), glutamic acid (Glu), histidyl (His),homophenylalanyl (HPhe), phenylalanyl (Phe), ornithyl (Orn), seryl (Set)and threonyl (Thr), and optionally substituted analogues thereof such asthiazoles and amino thiazoles. Preferably the side chain on the P₂ aminoacid is selected so that the linked P₂ amino acid is a member of thegroup consisting of phenylalanyl (Phe), leucyl (Leu), tyrosyl (Tyr) andvalyl (Val) amino acid residues and substituted analogues thereof,particularly including Tyr(OMe).

More specifically regarding the selection of side chains, the cathepsinsand the calpains share great cross reactivities with many inhibitors ofstructures shown above, although Cathepsin B responds most strongly tobasic side chains at P₁ (although reacting to several), while CathepsinL is more susceptible to neutral side chains at P₁. Both Cathepsin B andCathepsin L require neutral side chains at P₂. Cathepsins H and C preferto attach to unblocked peptides, with Cathepsin H favoring a singlepeptide, Cathepsin C a dipeptide and the calpains susceptible to neutralside chains. The cathepsins, as a general rule, are more reactive thanthe calpains. Interestingly, neither of these two enzyme types isinhibited when Asp occupies the P₁ position. In contrast, theinterleukin-1β-converting enzyme (ICE) is unaffected by all theseinhibitors unless Asp is at the P₁ position. This fundamental differencebetween the ICE enzyme and its inhibitors on the one hand, and the othercysteine enzymes and their inhibitors on the other, is well documentedin the literature.

When an aspartyl side chain is present in inhibitors based on anactivated ketone an unnatural event occurs--the free acid in the sidechain attacks the ketone (whose counterpart in the natural substrate isan unreactive amide carbonyl) and a thermodynamically favored hemiketalresults. Such hemiketals may be transition state mimics that are alsoknown to play a role in protease inactivation. The inhibition of the ICEenzyme can now follow either of two paths: hemiacetal exchange orthiolate attack on unmasked ketone, thus leading to some confusion inthe detailing of the mechanism of inhibition. The problem is eliminatedby esterification of the side chain.

One optimum peptide sequence for ICE inhibitors is known to be: B -Tyr - Val - Ala - Asp - Trap; where B is the blocking group and the"trap" is the activated ketone or aldehyde (reversible inhibitor). Dollehas shown that this sequence can be reduced to Val - Ala - Asp - andeven Asp alone is inhibitatory. Dolle et al., P₁ Aspartate--BasedPeptide α-((2,6-Dichlorobenzoxy)oxy)methyl Ketones as PotentTime-Dependent Inhibitors of Interleukin-1β-Converting Enzyme, 37 J.Med. Chem. 563.

The leaving groups of the present invention share certain features toassure the low toxicity and good solubility of the inhibitors. Inparticular, the leaving groups of the present invention: (1) immitate orimprove upon the cleaved peptide portion of the proteases naturalsubstrate; (2) activate the carbonyl of the inhibitor to selectivelyreact with the thiolate of a cysteine protease; (3) are non-toxic andnon-cleavable by non-cysteine proteases and esterases; and (4) are verywater soluble and enable the use of more amino acids than prior artleaving groups allow.

As indicated, the inhibitors of the present invention immitate orimprove upon the cleaved peptide portion of the proteases naturalsubstrate. The natural substrate leaving group is the sum of planar (ornearly so) amide bonds fused by tertiary substituted chiral carbonatoms. In applicant's prior application (Ser. No. 08/164,031) it wasdisclosed that oxygen fused to a heterocyclic ring could imitate theplanar features of the natural substrate leaving groups, and also that aheterocycle unit provides the diversity needed to imitate the differentelectronic and spacial specificity requirements of different individualenzymes. It was also demonstrated that the degree of aromaticity of thering was not a requirement for efficacy.

The inhibitors of the present invention activate the carbonyl of theinhibitor to selectively react with the thiolate of a cysteine protease.This concept was not appreciated until the demonstration of the successof peptidyl fluoroketones by Rasnick et al. in U.S. Pat. No. 4,518,528.The best replication of the chemistry ascribed to the mostelectronegative atom is to replace fluorine with the second mostelectronegative atom (oxygen) deshielded further by the attachment of anatom with electron withdrawing double bond character. The inhibitors ofthis invention maximize this premise by electronically coupling theanion of the leaving group to the electo positive center of a carbonylcarbon. By using a hydrocarbon structure devoid of halogens we eliminatethe toxicity associated with peptidyl fluoroketones, trifluoromethylsubstitutions, and halogenated hydrocarbons which are common to otherinhibitors in the art.

The inhibitors of the present invention are non-toxic and non-cleavableby non-cysteine proteases and esterases. In an attempt to minimizedipole moments, 1,3-dicarbonyls form very stable enols, and as a resultthe α-ketoethers prepared in this invention show outstanding stabilityand oral efficacy. On the other hand, 1,3-dicarbonyls are readilyeliminated through the Krebs Cycle and therefore pose less of a toxicitypotential than nitrogen aromatic heterocycles and other aromatics thatrequire liver oxidative clearance.

The inhibitors of the present invention are preferably very watersoluble and enable the use of more amino acids than current art leavinggroups in the peptide construction of the inhibitor. One generalizationthat can be made about the state of the art inhibitors is that theleaving group is of high molecular weight (as a dichlorophenol) whichreduces the overall water solubility and oral efficacy of the peptideinhibitor. The smaller more polar oxyheterocycles of this inventionactually increase the water solubility of the peptide inhibitor as shownin Example 8 where the leaving group derived from tetronic acidincreases the solubility of the peptide inhibitor almost two fold overthat of the peptide portion alone (approximated by thefluoroderivative). Further additions of hydroxyl groups to the parenttetronic nucleus further enhances water solubility and leaving groupssuch as those derived from ascorbic acid becomes most preferable.

Reference will now be made to specific examples for making and using thecysteine protease inhibitors of the present invention. It is to beunderstood that the examples are provided to more completely describepreferred embodiments, and that no limitation to the scope of theinvention is intended thereby.

EXAMPLE 1

N-Morpholinecarbonyl-L-phenylalanyl-L-homophenylalanine-α-(4-oxy-5-phenyl-4-cyclopentene-1,3-dione)methyl ketone.

N-morpholinecarbonyl-L-phenylalanyl-L-homophenylalanine bromomethylketone (100 mg, 0.194 mmol), potassium fluoride (45 mg, 0.775 mmol), and4-hydroxy-5-phenyl-4-cyclopentene-1,3-dione was placed in a 20 cm testtube equipped with a stirring bar and placed under an argon atmosphere.Next 3 ml of dry DMF was syringed into the reaction which was allowed tostir at room temperature until TLC (silica gel, CHCl₃ /isopropanol:95/5)showed total loss of starting material. The reaction was then passedthrough a short plug of silica gel (ethyl acetate) and the solvent wasremoved in vacuo. The resulting material was purified by size exclusionchromatography (LH 20, methanol) and precipitated in ether to give ayellow powder after filtration. (m.p. =155°-157° C., IC₅₀ Cathepsin B,94 nM.)

EXAMPLE 2

N-Morpholinecarbonyl-L-phenylalanyl-L-homophenylalanyl-α-(4-ascorbityl)methyl ketone.

N-morpholinecarbonyl-L-phenylalanine bromomethylketone (495 mg, 1 mmol),sodium ascorbate (380 mg, 2 equivalents), and potassium fluoride (116mg, 2 equivalents) was placed in a 50 mL round bottom flask under anatmosphere of argon. Next, 5 ml of dry DMF was syringed in and thereaction was allowed to stir at room temperature overnight. The next daythe reaction was filtered through celite and the solvents were removedin vacuo. The residue was dissolved in chloroform and the resultingsolution was diluted with an equal volume of methylene chloride toprecipitate the unreacted sodium ascorbate. After filtration the solventwas removed in vacuo and the residue purified by size exclusionchromatography to give a white solid, mp 105°-110° C. IC₅₀ Cathepsin B,141 nM.

In a similar manner the following compounds were prepared:N-Morpholinecarbonyl-L-phenylalanyl-L-homophenylalanyl-α-(4-oxy-6-methyl-2-pyrone)methyl ketone (m.p. 94°-98° C.);N-Morpholinecarbonyl-L-phenylalanyl-L-homophenylalanyl-α-(4-oxy-5,6-dihydro-6-methyl-2H-pyran-2-one)methyl ketone (m.p. 74°-78° C.);N-Morpholinecarbonyl-L-Leucyl-L-homophenylalanyl-α-(4-oxy-(6-methyl-2-pyrone)methyl ketone (m.p. 70°-75° C.);N-Morpholinecarbonyl-L-phenylalanyl-L-homophenylalanyl-α-(4-oxy-coumarin)methyl ketone (m.p. 115°-119° C.);N-Morpholinecarbonyl-L-phenylalanyl-L-lysyl-(4-oxy-(6-methyl-2-pyrone)methyl ketone (m.p. 151°-155° C.); N-Morpholinecarbonyl-L-tryosyl(O-methyl)-L- lysyl-(4-oxy-(6-methyl-2-pyrone) methyl ketone (m.p.140°-142° C.);N-Morpholinecarbonyl-L-phenylalanyl-L-homophenylalanyl-α-(4-oxy-3-phenyl-dihydrofuran-2-one)methyl ketone (m.p. 114°-116° C.); N-Morpholinecarbonyl-L-tryosyl(O-methyl)-L-lysyl-(4-oxy-3-phenyl-dihydrofuran-2-one) methyl ketone(m.p. 140°-142° C.);N-Morpholinecarbonyl-L-phenylalanyl-L-lysyl-α-(4-oxy-3-phenyl-dihydrofuran-2-one)methyl ketone (m.p. 140°-145° C.);N-Morpholinecarbonyl-L-phenylalanyl-L-homophenylalanyl-α-(4-oxy-dihydrofuran-2-one)methyl ketone (m.p. 155°-157° C.).

Structural formulas and in vitro activities (against Cathepsin B) forthese compounds are set forth below:

    ______________________________________                                        In Vitro Activity Against Purified Cathepsin B                                Compound                 IC.sub.50 Cat B                                      ______________________________________                                               ##STR22##             94 nM                                                   ##STR23##             141 nM                                                  ##STR24##             112 nM                                                  ##STR25##             567 nM                                                  ##STR26##             400 nM                                                  ##STR27##             45 nM                                                   ##STR28##             25 nM                                                   ##STR29##             83 nM                                                   ##STR30##             36 nM                                            10.                                                                                  ##STR31##             8 nM                                                    ##STR32##             5 nM                                                    ##STR33##             10 nM                                            ______________________________________                                    

EXAMPLE 3

Protocol for the in vitro evaluation of inhibitors with cathepsin B.

Enzyme: Cathepsin B, purified from human liver, is from Enzyme SystemsProducts (Dublin, Calif.). The activity is 50 mU per ml at 30° C., in 52mM sodium phosphate, pH 6.2, 31 mM DTT, 2.1 mM EDTA, with 0.2 mMZ-Arg-Arg-7-amino-4-trifluoromethyl-coumarin as a substrate. Specificactivity is 8330 mU per mg protein. (1 mU=1 nmol per min.)

Substrate Boc-Leu-Arg-Arg-7-amino-4-trifluoromethyl-coumarin-2HBr isfrom Enzyme Systems Products (Dublin, Calif.) and is known to be aspecific substrate for cathepsin B. A 20 mM solution is made in DMF andstored at 20° C.

Candidate inhibitors are dissolved in DMF and diluted to 20 mM andstored at 20° C. Dilutions are made in assay buffer.

The percent inhibition and the inhibitor concentration at which theenzyme is 50% inhibited (IC₅₀) are determined as follows. Five μl ofassay buffer (50 mM potassium phosphate pH 6.2, 2 mM EDTA, 5mM DTT) onice for 30 min. The inhibition is initiated by the addition of 5 ml of200 mM, 20 mM, and 2 mM inhibitor each to the 480 μl aliquots. The 485μl aliquot with enzyme is used as a control and thus receives noinhibitor. The enzyme/inhibitor mixtures are incubated 10 min. on iceand assayed for cathepsin B activity as follows: Cathepsin B assay: To490 μl of pre-incubated inhibitor/enzyme mixtures in assay buffer in 0.5ml cuvette at 37° is added 10 μl of the substrate. Final inhibitorconcentrations become 2000 nM, 200 nM, and 20 nM for the 200 μM, 20 μMand 2 μM stock concentrations, respectively. Activity is followed byrelease of free AFC over 5 min. The change in fluorescence is(fluorescence units at t=6)--(fluorescence units at t=1) with aPerkin-Elmer LS-5B spectrofluorometer (ex=400 nm, em=505 nm). Thepercent inhibition is determined by comparing the change in fluorescenceunits of the three sample concentrations of inhibited enzyme to thechange in fluorescence units of the control enzyme. The percentinhibition is calculated as:

100-(fl. units of sample/fl. units of control×100).

The IC₅₀ is ascertained by plotting percent inhibition vs. inhibitorconcentration on the log scale. The IC₅₀ is the concentration of theinhibitor (nM) at which the enzyme is inhibited by 50%.

IC₅₀ values for preferred inhibitors are shown on the table ofstructural formulas supra.

EXAMPLE 4

                  TABLE 1                                                         ______________________________________                                        Malarial Cysteine Protease Inhibition: IC.sub.50 Concentrations.sup.1         New Inhibitor                                                                               P. falciparum                                                                           P. vinckei                                            ______________________________________                                        6.           5-10 nM    5-10 nM                                               7.           ˜50 nM                                                                             ˜100 nM                                         9.           300-500 pM <1 nM                                                 11.          5-10 nM    ˜10 nM                                          12.          ˜10 nM                                                                             ˜10 nM                                          ______________________________________                                         .sup.1 The IC.sub.50 is the concentration of the inhibitor (nM) at which      the enzyrne is inhibited 50% within 6 minutes in our standard in vitro        assay (see Example 3)                                                    

Proteolytic activity assays. Gelatin-substrate PAGE is performeddescribed in Rosenthal, McKerrow, Rasnick, and Leech, Plasmodiumfalciparum: Inhibitors of Lysosomal Cysteine Proteinases Inhibit aTrophozoite Proteinase and Block Parasite Development, 35 Mol. Biochem.Parasitol. 177-184 (1989). In brief, this technique involveselectrophoresis of nonreducd proteins on a gelatin-containing gel,removal of SDS from the gel by washing with 2.5% Triton-100, overnightincubation (0.1M sodium acetate, 10 mM dithioerythritol (pH 6.0, 37° C.)of the gel to allow hydrosis of the gelatin by renatured proteinases,and staining with Coomassie blue. Proteinases are identified as clearbands in the blue staining gel. To evaluate the effects of proteinaseinhibitors, the inhibitors are incubated with parasite extracts (1 hr,room temperature) before samples are mixed with the electrophoresissample buffer, and they are included in the overnight gel incubationbuffer. Proteolytic activity was also measure with two other substrates:(a) fluorogenic peptide substrates containing the7-amino-4-methyl-coumarin (AMC) detecting group (Enzyme SystemsProducts, Dublic, Calif.) and (b) ¹⁴ C!-methemoglobin (Dupont NewEngland Nuclear, Wilmington, Del.), both as described in Rosenthal,McKerrow, Aikawa, Nagasawa, and Leech, A Malarial Cysteine Proteinase isNecessary for Hemoglobin Degradation by Plasmodium Falciparum. 82 J.Clin. Invest, 1560-66.(1988).

In another test of malarial inhibition by the cysteine proteaseinhibitors of the present invention compound A2, infra, was particularlyeffective.

EXAMPLE 5

                  TABLE 3                                                         ______________________________________                                        Inhibition of T. cruzi in Infected Cells with New Inhibitors.                             Survival Time                                                     Compound        Cell line J774                                                                            Cell line BHK                                     ______________________________________                                        Control          4 Days      5 Days                                           Mu Phe HPheCH.sub.2 F                                                                         16 Days plus                                                                              16 Days plus                                      6.              16 Days plus                                                                              16 Days plus                                      9.               4 Days      6 Days                                           ______________________________________                                    

Survival time is measured in days before the cell monolayer is destroyedby the infection. Irradiated BHK and J774 cells (six well plates) wereinfected with T. cruzi and simultaneously treated with 20 μM (3 ml totalvolume) with daily change of culture medium+plus inhibitor).

Cultivation and preparation of T. cruzi. Cloned and uncloned populationswere derived from the strains Brasil and CA-I and are cryopreserved inliquid nitrogen. Axenically cultured epimastigotes are maintained inexponential growth phase by weekly passage in Brain HeartInfusion-Tryptose medium (BHT media as given in Cazzulo, Cazzulo,Martinez, and Cazzulo, Some Kinetic Properties of a Cysteine Proteinase(Cruzipain) from Trypanosoma Cruzi. 33 Mol. Biochem. Parasitol. 33-42(1990)), supplemented with 20 μg/ml and 10% (v/v) heat inactivated fetalcalf serum (FCS). Different host cell lines (J774 mouse macrophage, BHK,etc.) are cultured with RPMI-1640 supplemented with 5% FCS at 37° in ahumidified atmosphere containing 5% CO₂. Trypomastigotes liberated fromthe host cells are used to infect new cultures for serial maintenance ofthe parasite in cell culture. The protocols used for the in vitro assaysof cysteine protease inhibitors are essentially as described by Harth,Andrews, Mills, Engel, Smith, and McKerrow, Peptide-Fluoromethyl KetonesArrest Intracellular Replication and Intercellular Transmission ofTrypanosoma Cruzi. 58 Mol. Biochem. Parasitol. 17-24 (1993), with theexception that in some experiments, the host cells are irradiated (2400RADs) before infection to prevent them from dividing.

EXAMPLE 6

In vitro inhibition of Pneumocystis carinii

When the following compounds were tested in a Pneumocystis cariniiculture system with human embryonic lung fibroblast monolayers, theorganism proliferation was inhibited as demonstrated below. The percentof inhibition is calculated as 100-(the number of P. cariniitrophozoites in a treated cell culture/number of trophosites of thecontrol)×100.

    ______________________________________                                               Percent inhibition                                                     Compound Day 1    Day 3       Day 5 Day 7                                     ______________________________________                                        Control  0        0           0     0                                         3        23       44          39    13                                        9        17       67          64    65                                        ______________________________________                                    

Methods. The drugs dissolved in dimethyl sulfoxide were diluted toconcentrations of 10 μg/ml for compound 3 and 10 μM/ml for compound 9 inminimum essential medium used for the culture of human embryonic lungfibroblasts. The final maximum dimethyl sulfoxide concentration was0.1%, a concentration of dimethyl sulfoxide that did not affect P.carinii proliferation when it is used alone and that gave P. cariniigrowth curves comparable to those of organisms in untreated controlwells. Cell cultures in 24-well plates were innoculated with P. Cariniitrophozoites (final concentration, about 7×10⁵ per ml) obtained frominfected rat lungs. Each culture plate contained untreated and treatedwells. Plates were incubated at 35° C. in a gas mixture of 5% O₂, 10%CO₂, 85% N₂ for up to 7 days. Plates were sampled on days 1, 3, 5 and 7by removal of 10 μl amounts after agitation of the cultures. The sampleswere placed on slides in 1-cm² areas, fixed in methanol and stained withGiemsa stain; and then they were examined microscopically as unknowns bytwo individuals. For each parameter there were four wells, making eightvalues for each parameter. Standard errors range from 3-13%. Cultureswere spiked with fresh drugs on days 2, 4 and 6.

EXAMPLE 7

The in vivo inhibition of cathepsin B in rat liver

    ______________________________________                                                 Time Post Dose (Hours)                                               Compound    1.5    3        6     12     24                                   ______________________________________                                        (via stornach tube).                                                           6          48     29       N/A   30     0                                    12                 53       32                                                (1600 nM dose)                                                                12                 35       31                                                (800 nM dose)                                                                 (via injection, IP)                                                           12                 79       70                                                (1600 nM dose)                                                                12                 55       70                                                (800 nM dose)                                                                 ______________________________________                                    

Protocol for the in vivo Evaluation of Inhibitors. Female Sprague Dawleyrats (150-200g each) are purchased from Simonson, Gilroy, Calif. After 1week of acclimation in-house, the animals (usually four per group) aredosed by the selected route of administration. Test compounds aredissolved in ethanol and diluted to the appropriate concentration withwater. In control studies, animals are dosed only with ethanol watervehicle.

Tissue Homogenate Preparation. At the appropriate time post dose, thetreated animals are anesthetized with ether, decapitated andexsanguinated. The tissues of interest are removed, quickly frozen inliquid nitrogen and then are stored at -70° C. until processing. Allsubsequent manipulations of the tissue samples are carried out at 4° C.Liver and skeletal muscle are pulverized while still frozen and thenhomogenized, while other target tissues are homogenized without priorpulverization. The tissue homogenization, in distilled water or 0.1%Brig-35, are subsequently performed using three 15-s bursts with a 10Nprobe on a Tekmar Tissuemizer set to 75-80% power. The samples arecentrifuged at 15000 g for 40 min; they partition into a lipid layer, alower clarified layer and a solid pellet. The clarified supernatant iscarefully aspirated and transferred to clean polypropylene tubes forstorage at -70° C., until the fluorometric assay for enzyme activity canbe performed.

Purified Lysosomal Enzyme Preparation. The procedure is based on areport by Bohley et al. (1969) and Barrett and Kirshke (1981). At theappropriate time post dose, the treated animals are anesthetized withsodium barbital and the livers are perfused in situ with ice-coldsaline. The livers are then removed, rinsed with ice-cold saline,blotted and weighed. The animals are sacrificed with ether. Allsubsequent manipulations of the tissue samples are carried out at 4° C.The livers are homogenized in 2 volumes of 0.25M sucrose at 0° C. with a30 ml Wheaton Teflon-on-glass homogenizer, using five full strokes witha motor setting at 55. Following centrifugation at 600 g for 10 minutes,the supernatant is transferred to clean tubes for centrifugation at 3000g for 10 minutes. The resulting supernatant is centrifuged for 15minutes. The lysosomal pellet is washed twice with 0.25M sucrose, lysedin 2.5 volumes of distilled water using a glass-on-glass homogenizer,and then centrifuged at 1000 g for 60 minutes. The supernatant is storedat -70° C. until fluorimetric assay for enzyme activity is performed.

EXAMPLE 8

Aqueous solubilities ofMorpholinecarbonyl-phenylalanyl-homophenylalanyl-α-(4-oxy-dihydrofuran-2-one)methyl ketone vs.Morpholine-carbonyl-phenylalanyl-homophenylalanyl-fluoromethylketone.

The aqueous solubilities of the two title compounds at 20° C. weredetermined by using UV spectroscopy and compared to that of a knownstandard benzoxycarbonyl-phenylalanyl-alanylfluoromethyl ketone. Theaqueous solubility of Mu-Phe-HPhe-α-(4-oxy-dihydrofuran-2-one) methylketone 12 was measured to be 0.277 mg/ml. The aqueous solubility ofMu-Phe-HPhe-CH₂ F was measured to be 0.140 mg/ml. The aqueous solubilityof Z-Phe-Ala-CH₂ F was 0.045 mg/ml at 14° C.

Experimental. A saturated solution was prepared by weighing 10 mg of thematerial and placing it in 5 ml of distilled and deionized water. Thesolution was capped and stirred at 20° C. unless otherwise noted. A 1 mlaliquot was removed after 24 hours and was filtered through a 0.45 μmfilter and diluted 1:50 with distilled and deionized water. Subsequentaliquots were taken and similarly diluted after 48, 72, and 96 hoursrespectively. The absorbances were measured at 247 nm for compound 12and at 219nm for Mu-Phe-HPhe-CH₂ F and were compared against a series ofrespective standard solutions run under similar conditions.

EXAMPLE 9

Synthesis of the cathepsin H inhibitors.

L-Homophenylalanyl-α-(4-oxy-(6-methyl-2-pyrone) methyl ketone.BOC-homophenyl-bromomethylketone (300 mg), potassium fluoride (195 mg),potassium carbonate (233 mg) and 4-hydroxy-6-methyl-2-pyrone (212 mg)was placed in a round bottom flask under an atmosphere of argon. Aboutone mL of DMF was added and the mixture was stirred at 50° C. for 40min. The reaction was then diluted with ethyl acetate (10×) and passedthrough a plug of silica gel to remove the salts. The solvents wereremoved under vacuum. The BOC-protecting group was removed by dissolvingthe resulting solid in 3 mL of methylene chloride and adding 3 mL of 4NHCI-dioxane. The reaction was run until only a stationary spot wasdetected with silica gel TLC (9:1, CHCl₃ :isopropanol). The resultingmixture was then added dropwise to 50 mL of ether and the precipitatedsolid was filtered. mp. 177°-179° C. IC₅₀ Cathepsin H: 118 nM.

In the same manner L-Homophenyl-α-(4-oxy-dihydrofuran-2-one)methylketone hydrochloride was synthesized. mp. 128°-132° C. IC₅₀ Cathepsin H:251 nM.

Numerous other cathepsin H inhibitors can be made with the constructionof an unblocked amino acid on an α-oxy heterocycle methyl ketone.

EXAMPLE 10

SYNTHESIS OF ICE INHIBITORS

The following example is meant to be illustrative but is not meant to berestrictive to other variations which would involve exchanges ofblocking groups, abbreviation or minor alterations in side chainconstruction, or exchanges with other leaving groups of this invention.

N-Benzoxycarbonyl-valyl-alanyl-aspartyl-α-(4-oxy-(6-methyl-2-pyrone)methyl ketone. Z-Val-AlaOMe: N-Benzoxycarbonyl-valine was dissolvedunder argon in 300 mL of freshly distilled THF and the resultingsolution was cooled in a methanol-ice bath. One equivalent of N-methylmorpholine followed by one equivalent of isobutylchloroformate was addedand the reaction was allowed to activate for 20 minutes. Anotherequivalent of N-methylmorpoline is then added followed by one equivalentof solid alanine methyl ester hydrochloride salt. The reaction wasallowed to come slowly to room temperature and stir overnight. The nextday the reaction was poured into 200 mL of 1N hydrochloric acid andextracted with ethyl acetate (2×150 mL). The combined organic fractionswere washed with brine (50 mL), aqueous sodium bicarbonate (100 mL),dried over MgSO₄, filtered and the solvents were removed under reducedpressure to give 14 g of a white solid methyl ester. mp. 157°-163° C.

Hydrolysis of the methyl ester was effected by dissolving 2.20 g in 35mL of methanol followed by the addition of 8.2 mL of 1N aqueous sodiumhydroxide solution. The reaction was stirred at room temperature for 4hours. At this time TLC (silica gel/CHCl₃ /isopropanol) showed that mostof this material had been converted to the acid (stationary spot onTLC). The methanol was then removed under reduced pressure and theresidue was dissolved in water (100 mL) an additional 5 mL of sodiumhydroxide was added and the water was washed with ethyl acetate (50 ml)and then neutralized with 1N hydrochloric acid, and extracted with 2×100ml of ethyl acetate. The organic layer was dried over MgSO₄, filteredand the solvents were evaporated to give a white solid: mp. 170°-175° C.

Condensation with aspartyl (O-t-butyl)-O-methyl ester was effected asfollows: Z-Val-Ala-OH was dissolved in 300 mL of freshly distilled THFand the resulting solution was cooled in an ice-methanol bath. Next oneequivalent of N-methyl morpholine was added followed by one equivalentof isobutylchloroformate and the reaction was allowed to activate for 20minutes. Another equivalent of N-methyl morpoline was added followed byone equivalent (5 g) of HCI-Asp(OtBu)OMe. The mixture was allowed tocome slowly to room temperature and stir overnight. The next day thereaction was poured into 200 ml of 1N hydrochloric acid and extractedwith ethyl acetate. The organic layer was washed with sodium bicarbonate(aq, 50 mL), brine (50 mL) and the organic layer was dried over MgSO₄,filtered, and the solvents were removed under reduced pressure. Theresidue was crystallized from 50 mL of methylene chloride and 200 mL ofether to give white crystals (4.0 g), mp. 157°-163° C.

Hydrolysis to Free Acid: Z-Val-Ala-Asp(otBu)OMe (4.6 g) was dissolved in30 mL methanol and then 12 mL of 1N sodium hydroxide (aq) was added andthe reaction was stirred for 1 hour at room temperature. At the end ofthis time the methanol was removed under reduced pressure and 50 ml ofwater plus another 12 mL of 1N solium hydroxide was added to dissolvethe precipitated solid. The resulting water solution was washed withethyl acetate (50 mL) and then the water fraction was acidified with 1NHCI and the resulting mixture extracted with ethyl acetate, dried overMgSO₄, filtered and concentrated to give 3.74 g ofZ-Val-Ala-Asp(O-tBu)OH.

Conversion to the Diazoketone: Z-Val-Ala-Asp(O-tBu)OH was dissolved in200 mL of freshly distilled THF and a methanol-ice bath was applied.Next one equivalent of N-methyl morpholine followed by one equivalent ofisobutyl chloroformate was added and the reaction was allowed toactivate for 20 minutes and then the resulting mixture was pouredthrough filter paper into diazomethane/ether made from 6.3 g of Diazald(Aldrich) according to the supplier's directions. The reaction wasallowed to stand overnight and then worked up in the following way: Thereaction was washed with water (2×50 mL), sodium bicarbonate (50 mL),brine (50 mL) and then dried over MgSO₄, filtered and the solvents wereremoved under reduced pressure to give a yellow solid 3.75 g. Thisresidue was then chromatographed in two parts through a 1×12 inch silicagel (CHCL₃ :isopropanol/95:5) column to give two product fractions. Theproduct with the lower R_(f) value (0.3) was shown by the absorption at5.54 ppm in the 100 MHz NMR to be the correct product.

Conversion to the Bromoketone: Z-Val-Ala-Asp(OtBu)CHN₂ was dissolved in25 mL ether and 25 mL THF and a methanol-ice bath was applied. Next 0.1mL HBr/acetic acid (30%) diluted to 10 mL with ether:THF (1:1) was addeddropwise. The yellow solution becomes clear and when no more colorremains the reaction is poured into an equal volume of brine, theorganic layers are separated and the water fraction is washed with anadditional 50 mL THF:ether. The organic fraction was then washed with 50mL of aqueous sodium bicarbonate, 50 mL brine, dried over MgSO₄,filtered and concentrated to give a white solid: mp. 150°-151° C.

Conversion to the α-(4-oxy-(6-methyl-2-pyrone) methyl ketone:Z-Val-Ala-Asp(otBu)CH₂ Br (131 mg), 4-hydroxy-6-methyl-pyrone (58 mg, 2equivalents), potassium fluoride (53 mg), and 1.5 mL of DMF was stirredat room temperature for two hours ah which time TLC (silica gel, CH₃Cl/isopropanol:97/3) showed loss of starting material and development ofproduct. The reaction was then run through a plug of silica gel (CHCl₃/isopropanol/9:1) and the solvents were removed under reduced pressure.Most of the excess pyrone starting material was precipitated fromisopropyl ether: CH₂ Cl₂ and theZ-Val-Ala-Asp(OtBu)-α-(4-oxy-6-methyl-pyrone) methyl ketone was isolatedfrom the resulting mother liquor by removal of the solvent and sizeexclusion chromatography: NMR (100 MHz, CDCl₃) δ 0.9 (dd, 6), 1.4 (broads+d, 12), 2.1 (s, 3), 3.5 (s, 2), 5.1 (s, 2), 7.3 (m, 5).

Removal of the side chain t-butyl group:

Z-Val-Ala-AsP(OtBu)CH₂ O-(6-methyl-pyrone) was dissolved under argon in2 ml of methylene chloride add 2 mL of 50% trifluoroacetic acidmethylene chloride was added and the resulting clear solution wasstirred for 30 minutes. At this time silica gel TLC (CHCl₃/isopropanol:9/1) showed loss of starting material (starting materialR_(f) 0.66; product R_(f) 0.44). The reaction was diluted twofold withchloroform and the solvents and reagents removed under reduced pressureto give a white solid, mp. 158°-163° C. (with multiple phase changesprior to melting).

Synthesis of N-Morpholinecarbonyl-L-Valyl-L-Alanyl-Aspartyl(OtBu)-α-(ascorbityl) methylketone. N-Morpholinecarbonyl-L-aline methylester: HCL-Valine methyl ester (25 g) was dissolved under argon in 600mL of freshly distilled THF and 100 mL of dry DMF and 1.0 equivalents ofN-methyl morpholine. The resulting solution was cooled to -15° and anadditional 1.1 equivalent of N-methyl morpholine followed by 1.1equivalents of morpholine chloride was added. The reaction was allowedto come slowly to room temperature and stir overnight. The reaction isthen poured into 300 mL of 1N HCL and extracted with ethyl acetate(2×200 mL). The combined organic fractions were washed with 1N HCL (50mL), brine (50 mL), dried over MgSO₄, filtered and the solvents wereremoved under reduced pressure to give 33 g ofN-morpholinecarbonyl-valine methyl ester as a white solid: NMR (100 MHz,CDCl₃ δ 0.9 (dd, 6), 2.1 (septet, 1), 3.0 and 3.65 (morpoline triplets,4 and 4), 4.98 (N-H).

Conversion to Mu-Val-OH: The above methyl ester was dissolved in 300 mLof methanol and 157 mL of 1N aqueous sodium hydroxide was added. Thereaction was stirred at room temperature for 2 hrs. after which time TLCshowed the product as a stationary spot. The methanol was removed underreduced pressure and an additional 28 mL of 1N aqueous sodium hydroxidewas added and the water fraction was washed with ethyl acetate (75 mL).The water fraction was then acidified with 185 mL of 1N HCl, 3/4 of thewater was removed under reduced pressure and the resulting mixture wasextracted with ethyl acetate (2×300 mL). The organic fraction was washedwith 1N HCl (50 mL), brine (50 mL), dried over MgSO₄, filtered andconcentrated to give N-morpholinecarbonyl-valine 29.75 g (78% yield).

Condensation with Alanine methyl ester: Mu-Val-OH was dissolved in 300mL of freshly distilled THF under argon and the solution was cooled to-15° C. Next one equivalent of N-methyl morpholine followed by oneequivalent of isobutylchloroformate was added. The reaction was allowedto activate 20 minutes and then another equivalent of N-methylmorpholine followed by alanine methyl ester hydrochloride salt wasadded. The reaction was allowed to come slowly to room temperature andto stir overnight. The next day the reaction was poured into 1Nhydrochloric acid and extracted with ethyl acetate (2×200 mL). Thecombined organic layers were washed with 1N hydrochloric acid (50 mL),brine, dried over MgSO₄, filtered, and the solvents were removed underreduced pressure to give 10.74 g (78%) ofN-morpholinecarbonyl-valyl-alanyl methyl ester. NMR (100 MHz, CDCl₃) δ1.4 (d, Ala CH₃), 3.7 (s, OMe) ppm.

Hydrolysis to the Free Acid: Mu-Val-Ala-OMe (1 g) was dissolved in 15 mLof methanol and then 4.8 mL of 1N sodium hydroxide (aq) was added. Thereaction was allowed to continue until TLC (CHCl₃ /isopropanol:9/1)showed only a stationary spot. The methanol was then removed underreduced pressure and an additional 1.2 mL of ethyl acetate and the waterfraction was acidified with 6 mL of 1N HCl. The mixture is extractedwith about 50 mL of ethyl acetate and the organic fraction washed with 5ml of 1N HCl, dried over MgSO₄, filtered and concentrated to give 0.8 g(84%) of a white solid which was characterized by the loss of the NMRabsorption at 3.7 ppm.

Condensation with Asp(OtBu)OH: Asp(OtBu)OH (2.51 g) was dissolved in 40mL of dry DMF under argon and 8.2 mL of bis(trimethylsilyl)acetamide(BSA) and the reaction was allowed to stir 40 minutes. In a separateflask, Mu-Val-Ala-OH (4.0 g) was dissolved in 200 mL of dry THF underargon and the resulting solution was cooled to -15° C. and oneequivalent of N-methylmorpholine was added followed by one equivalent ofisobutylchloroformate and the resulting mixture was allowed to stir 20minutes and then the first reaction was then poured into the secondreaction and both were maintained at -15° C. for one hour and thenallowed to come slowly to room temperature and to stir overnight. Thereaction was poured into 150 mL of 1N HCl and extracted with 2×200 mL ofethyl acetate. The combined organic fractions were washed with 15 mL 1NHCl, brine (50 mL), dried over MgSO₄ (with decolorizing carbon),filtered, and the solvents were removed under reduced pressure to yield4.89 g of Mu-Val-Ala-Asp(OtBu)OH.

Conversion to the diazoketone: Mu-Val-Ala-Asp(OtBu)OH (4.89 g) wasdissolved in 250 mL of freshly distilled THF under argon and theresulting solution was cooled to -15° C. Next one equivalent of N-methylmorpholine followed by one equivalent of isobutyl chloroformate wasadded. The reaction was allowed to activate at this temperature for 20minutes and then poured through a filter into a solution of diazomethanein ether that was made from 10.8 of diazald according to the supplier's(Aldrich) directions. The reaction was allowed to come slowly to roomtemperature and to stir overnight. The next day the reaction was washedwith water, bicarbonate and brine (50 mL each), dried over MgSO₄,filtered and the solvents were removed under reduced pressure to give ayellow oil showing five spots on TLC (silica gel, CHCl₃/isopropanol:97/3). The lowest R_(f) is isolated by chromatography on300 g of silica gel and is shown to be the product by the CHN₂absorption in the NMR at δ 5.75.

Conversion to the bromoketone: Mu-Val-Ala-Asp(OtBu)CHN₂ was dissolved in45 mL of methylene chloride and the resulting solution was cooled to-15° C. Next 1.7 ml of 30% methylene chloride dissolved in 30 mlmethylene chloride was added dropwise and the reaction was monitored byTLC (silica gel, CHCl₃ /isopropanol). The reaction was then poured intobrine and the organic fraction was washed with sodium bicarbonate (aq),brine, and dried over MgSO₄, filtered, and the solvents were removedunder reduced pressure to give a crude gold solid which was purified bydissolving the material in a minimum of methylene chloride andprecipitation in ether/hexane. The product Mu-Val-Ala-Asp(OtBu)CH₂ Br ischaracterized in the NMR (100 MHz, CDCl₃) by the disappearance of thediazoketone absorbance at δ 5.75 and the appearance of a singlet at δ4.18.

ICE Inhibitors: Mu-Val-Ala-Asp(OtBu)CH₂ Br (0.36 mmol), potassiumfluoride (1.09 mmol) and the hydroxy heterocycle (0.546 mmol) was sealedunder argon and then 8 mL of dry DMF was added and the reaction wasallowed to stir overnight. The next day the reagents were removed eitherby dilution with ethyl acetate and washing brine or by passage throughsilica gel. The solvents were removed under vacuum and the product wasisolated by size exclusion chromatography (LH20). In this manner thefollowing compounds were prepared:

N-Morpholinecarbonyl-L-Valyl-L-Alanyl-Aspartyl(OtBu)-α-(ascorbityl)methylketone (mp. 138°-144° C.);N-Morpholinecarbonyl-L-Valyl-L-Alanyl-Aspartyl(OtBu)-α-(-4-oxy-(3-azo-m-anisidine)methyl ketone: NMR (CDCl₃) δ 0.95 (dd, 6H, Val Ch₃), 1.4 (s, 12H,OtBu+Ala CH₃), 2.1 (m, 1H, Val CH), 2.9 (d, 2H, CH₂ sidechain), 3.3 (t,4H, MU), 3.7 (t, 4H, MU), 3.85 (s, 3H, OCH₃, 4.2 (t, 1H), 4.6 (t, 1H),4.7 (d, 2H), 4.9 (m, 3H, CH₂ O), 6.9 (d, 1H), 7.1 (m, 4H) 7.9 (d, 1H).

Removal of the tBu in the above inhibitors was effected with 25%trichloroacetic acid in methylene chloride to give the correspondingfree acid inhibitors.

EXAMPLE 11

SYNTHESIS OF CALPAIN INHIBITORS

The following example is meant to be illustrative of a calpain inhibitorbut is not meant to be restrictive as numerous variations in peptide andleaving groups of this invention can be envisioned without undueexperimentation.

Acetyl-Leucyl-Leucyl-Phenylalanyl-α-(-4-Oxy-dihydrofuran-2-one) methylketone. Ac-Leu-Leu-OCH₃ : Acetyl-Leucine (5.0 g) was dissolved in 150 mLof distilled THF under argon and the resulting solution was cooled to-15°. Next one equivalent of N-methyl morpholine followed by oneequivalent of isobutyl chloroformate was added and the reaction wasallowed to activate 20 minutes and then another equivalent of N-methylmorpholine followed by HCl-LeuOMe (5.25 g). The reaction is allowed toslowly come to room temperature and stir overnight. The reaction wasthen poured into 150 mL of 1N HCl and extracted with ethyl acetate(2×150 mL). The combined organic fractions were washed with 1N HCl (15mL), brine (50 mL), and dried over MgSO₄, filtered and the solvent wasremoved under reduced pressure and then high vacuum. TLC (silica gel,CHCl₃ /isopropanol:95:5) showed the product Ac-Leu-Leu-OCH₃ to be asingle spot R_(f) 0.36. NMR (100 MHz) δ 0.95 (d, 12H), 1.5 (bs, 6H), 2.0(s, 3H), 3.7 (s, 3H), 4.5 (q, 2H), 6.6 (d, 1H), 6.8 (d, 1H).

Hydrolysis to the Free Acid: Ac-Leu-Leu-OCH₃ (7.8 g) was dissolved in150 mL of methanol and then 38 mL of 1N sodium hydroxide was added andthe reaction was allowed to stir at room temperature for about 4 hours.The methanol was removed under reduced pressure and an additional 10 mLof 1N sodium hydroxide was added and the water fraction was extractedwith 10 mL of ethyl acetate. The water fraction was then neutralizedwith 1N HCl and extracted with ethyl acetate (3×50 mL). The combinedorganic fraction was washed with brine and dried over MgSO₄, filteredand the solvent was removed under reduced pressure. δ 0.95 (d, 12H), 1.5(br s, 6H), 2.0 (s, 3H), 4.5 (q, 2H), 6.6 (d, 1H), 6.8 (D, 1H), 9.5 (bs,1H).

Condensation with PheOMe. Ac-Leu-Leu-OH was dissolved in 200 mL ofdistilled THF under argon and the solution was cooled to -15° C. Nextone equivalent of N-methyl morpholine followed by one equivalent ofisobutyl chloroformate was added and the reaction was allowed toactivate for 20 minutes. An additional equivalent of N-methyl morpholinefollowed by HCl-HPheOCH₃ was added and the reaction was allowed to comeslowly to room temperature and stir overnight. The reaction was pouredinto 200 mL of 1N HCl and extracted with ethyl acetate (2×150 mL). Theorganic fraction was washed with 1N HCl (20 mL), brine (50 mL), anddried over MgSO₄, filtered and the solvents removed under reducedpressure and then high vacuum to leave a solid white cake (TLC, silicagel, CHCl₃ /isopropanol R_(f) 0.35). NMR (100 MHz) 6 0.95 (d, 12H), 1.6(bs, 6H), 2.1 (s, 3H), 3.1 (d, 2H), 3.6 (s, 3H), 4.7 (m, 3H), 6.9 (d,1H), 7.2 (m, 5H), 7.5 (d, 1H).

Conversion to the Free Acid. Ac-Leu-Leu-Phe-OMe was dissolved in 150 mLof methanol and 26 mL of 1N sodium hydroxide was added and the rectionwas stirred 4 hours at which time the methanol was removed under reducedpressure and an additional 7 mL of sodium hydroxide was added. Thiswater fraction was then washed with ethyl acetate (10 mL) andneutralized by the addition of 1N HCL. The resulting mixture wasextracted with ethyl acetate 2×100 mL and the extract washed with 1NHCL, brine and dried over MgSO₄, filtered and the solvents were removedunder reduced pressure to give 7.56 g (94%) of Ac-Leu-Leu-Phe-OH as awhite solid. The NMR (100 MHz, CDCl₃) of the product acid bears strikingresemblance to that of the precursor ester except for the loss of asignal at δ 3.6 and the appearance of a broad singlet at 10.1 (1H).

Conversion to the Diazoketone: Ac-Leu-Leu-Phe-OH (4.68 g) was dissolvedin 200 mL of freshly distilled THF and a methanol-ice bath was applied.Next one equivalent of N-methyl morpholine followed by one equivalent ofisobutyl chloroformate was added and the reaction was allowed toactivate for 20 minutes and then the resulting mixture was pouredthrough filter paper into diazomethane/ether made from 10.8 g if Diazaldaccording to the supplier's (Aldrich) directions. The reaction wasallowed to come slowly to room temperature and to stand overnight. Thereaction was washed with water (2×50 ml), sodium bicarbonate (50 mL),brine (50 mL), and then dried over MgSO₄, filtered and the solvents wereremoved under reduced pressure to give after column chromatography(silica gel, CHCl₃ /isopropanol:93/7) 3.07 g (61%) of a yellow powder.NMR (100 MHz, CDCl₃) δ 0.95 (d, 12H), 1.6 (bs, 6H), 2.1 (s, 3H), 3.1 (d,2H), 4.7 (m, 3H), 5.6 (s, 1H), 6.9 (d, 1H), 7.2 (m, 5H), 7.5 (d, 1H).

Conversion to the Bromide. Ac-Leu-Leu-Phe-CHN₂ (1 g) was dissolved in175 mL of methylene chloride and then 1.2 mL of 30% HBr/acetic acid thathad been diluted with 25 mL methylene chloride was added dropwise at-15° C. As the reaction proceeds bubbles evolve with the formation of aprecipitate. The reaction was monitored by TLC (silica gel/CHCl₃-isopropanol: 9/1; R_(f) product 0.54). Upon completion, the rection waspoured into 150 mL of brine and the reaction flask was washed withanother 150 mL of methylene chloride to dissolve the residualprecipitate. The combined organic layers were washed with sodiumbicarbonate (aq, 50 mL), brine (50 mL), dried over MgSO₄ andconcentrated to give a dull white solid. This solid was purified byprecipitate from methylene chloride into ether to yield 610 mg (54%) ofa white solid TLC (silica gel, CHCl₃ /isopropanol:9:1) one spot R_(f)0.54. NMR (DMSO-d₆) δ 0.81 (d, 12H), 1.3 (m, 6H), 1.8 (s, 3H), 3.1 (d,2H), 4.1 (m, 2H), 4.3 (s, 2H), 4.6 (q, 1H), 7.2 (m, 5H), 8.0 (d, 2H),8.4 (d, 1H).

Calpain inhibitor: Ac-Leu-Leu-Phe-CH2Br (200 mg), tetronic acid (65 mg)and potassium fluoride (68 mg) were mixed under argon with 5 mL of dryDMF overnight. The reaction was then diluted with 20 mL of ethyl acetateand the reaction was washed with 10 mL sodium bicarbonate (aq), brine(10 mL), and dried over MgSO₄. The reaction was filtered and thesolvents were removed under reduced pressure and then high vacuum togive 107 mg (49%) ofacetyl-leucyl-leucyl-phenylalanyl-α-(4-oxy-dihydrofuran-2-one)methylketone.

EXAMPLE 12

SYNTHESIS OF OTHER HETEROCYCLIC INHIBITORS

Using the following procedures other heterocyclic cathepsin inhibitorsare prepared.

N-Morpholinecarbonyl-L-phenylalanyl-L-homophenylalanine-α-(4-oxy-N-acetyl-prolinemethyl ester) methyl ketone (A1). MuPheHPheCH₂ Br (250 mg),N-acetyl-proline methyl ester (2.0 g), potassium fluoride (232 mg), andpotassium carbonate (276 mg) are placed under argon and then 1.5 mL ofdry DMF was added and the reaction was allowed to stir at roomtemperature for 100 minutes. The reaction was then passed through ashort silica gel column (ethyl acetete) and the solvents were removed invacuo. Precipitation in ether produced a white solid, mp. 81°-84° C.

N-Morpholinecarbonyl-L-phenylalanyl-L-homophenylalanyl-α-(3-oxy-5-ethyl-4-methyl2(5H) furanone) methyl ketone (A2). MuPheHPheCH₂ Br (100 mg), potassiumfluoride (45 mg), and 5-ethyl-3-hydroxy-4-methyl-2(5H) furanone (110 mg)was placed under argon in 5 mL of dry DMF and the reaction was stirredat room temperature overnight. The next day the reaction was dilutedwith ethyl acetate and washed with aqueous sodium bicarbonate and thebrine, dried over MgSO₄, filtered and the solvents were removed invacuo. The product was purified by size exclusion chromatography (LH-20,methanol) to give a white solid, mp. 65°-71° C.

N-Morpholinecarbonyl-L-phenylalanyl-L-homophenylalanine-α-(8-oxy-quinoline)methyl ketone (A3). To MuPheHPheCH₂ Br (100 mg), potassium fluoride (45mg) and 8-hydroxyquinoline (123 mg) in a test tube under argon was added5 mL of dry DMF and the reaction was allowed to stir for four hours. Thereaction was then passed through a short silica gel column and thesolvents were removed in vacuo. The product was purified by first sizeexclusion chromatography (LH-20, methanol) and then by crystallizationfrom methylene chloride/ether to give 65 mg of crystals. The product wascharacterized by NMR (100 MHz) δ 8.5-8.0 (m, hetero aromatic), 7.5-6.5(mm, homo and heteroaromatic), 3.75-3.5, 3.25-3.0 (2 m, Mu H), 2.75 (s,heteroaromatic Me) ppm.

N-Morpholinecarbonyl-L-phenylalanyl-L-homophenylalanine-α-(2-oxy-4-methyl-quinoline)methyl ketone (A4). MuPheHPheCH₂ Br (100 mg), potassium fluoride (45mg), and 2-hydroxy-4-methyl-quinoline (123 mg) was placed in a test tubeunder argon and 5 mL of dry DMF was added. The reaction was allowed tostir at room temperature overnight. The reaction was then passed througha short silica gel plug and the solvents were removed in vacuo. Theresidue was purified first by size exclusion chromatography and then byprecipitation into ether to give a solid, mp. 180°-183° C.

N-Morpholinecarbonyl-L-phenylalanyl-L-homophenylalanine-α-(4-oxy-quinoline)methyl ketone (A5). MuPheHPheCH₂ Br (100 mg), potassium fluoride (45mg), and 4-hydroxyquinoline was placed in a test tube under argon and 5mL of dry DMF was added. The reaction was stirred for 3.5 hours and thenpassed through a short silica gel column (ethyl acetate). The solventswere removed in vacuo and the residue was purified by size exclusionchromatography followed by precipitation of the collected product inether Co yield a white powder, mp. 107°-111° C.

N-Morpholinecarbonyl-L-phenylalanyl-L-homophenylalanine-α-(4-oxy-quinazoline)methyl ketone (A6). MuPheHPheCH₂ Br (100 mg), 5-methyl-5-triazolo1,5a!-pyrimidin-7-ol (116 mg), and potassium fluoride (45 mg) was addedtogether under argon in a dry test tube and 5 mL of dry DMF was added.The reaction was stirred at room temperature for 3.5 hours and then thereaction was diluted with ethyl acetate and passed through a plug ofsilica gel. The solvents were removed in vacuo and the product waspurified by size exclusion chromatography (LH-20, methanol) to give asolid product, mp. 129°-132° C.

N-Morpholinecarbonyl-L-phenylalanyl-L-homophenylalanine-α-(2-oxy-benzimidazole)methyl ketone (A7). MuPheHPheCH₂ Br (100 mg), 2-hydroxybenzimidazole(104 mg), and potassium fluoride (45 mg) was added together under argonin a dry test tube and 5 mL of dry DMF was added. The reaction wasstirred at room temperature for 3 hours and then the reaction wasdiluted with ethyl acetate and passed through a plug of silica gel. Thesolvents were removed in vacuo and the product was purified by sizeexclusion chromatography (LH-20, methanol) to give an off white solidproduct, mp. 115°-120° C.

N-Morpholinecarbonyl-L-phenylalanyl-L-homophenylalanine-α-(1-oxy-isoquinoline)methyl ketone (A8). MuPheHPheCH₂ Br (100 mg), potassium fluoride (45mg), isocarbostyril (112 mg), are placed under argon and then 4 mL ofdry DMF is added. The reaction is stirred at room temperature for threehours and then the reaction is diluted with ethyl acetate and passedthrough a short silica gel column. The solvents are removed in vacuo andthe product purified by size exclusion chromatography (LH-20, methanol)to give a white solid, mp. 104°-107° C.

N-Morpholinecarbonyl-L-phenylalanyl-L-homophenylalanine-α-(7-oxy-coumarin)methyl ketone (A10). MuPheHPheCH₂ Br (200 mg) and potassium fluoride (90mg) was added under argon to 1.5 mL of DMF and 250 mg of7-hydroxycoumarin was added. The reaction turns a bright gold and isallowed to stir for one hour at which time TLC shows total loss ofbromide. The reaction was then passed through a short column of silicagel (CHCl₃ /isopropanol, 9:1) and the solvents were removed in vacuo.Further chromatography (LH-20/methanol) gave after removal of solvent awhite solid foam, mp. 87°-89° C.

In a like mannerN-Morpholinecarbonyl-L-phenylalanyl-L-homophenylalanyl-α-(7-oxy-4-methyl-coumarin)methyl ketone (A9) was prepared, mp. 99°-102° C.

N-Morpholinecarbonyl-L-phenylalanyl-L-homophenylalanine-α-(2-oxy-benzofuran)methyl ketone (A11). MuPheHPheCH₂ Br (100 mg), 2-coumaranone (52 mg) andpotassium fluoride (45 mg) were placed in a test tube under argon andthen one mL of DMF was added and the reaction turns a cherry red. Thereaction after 20 minutes shows a loss of starting bromide (TLC, Silicagel, CHCl₃ /isopropanol:9/1) R_(f) product, 0.59; R_(f) bromide 0.48.The reaction was passed through a short plug of silica gel (ethylacetate) and the solvents were removed in vacuo. The residue wasdissolved in a minimum of methylene chloride and precipitated in etherand the precipitate filtered to yield a white solid, mp. 94°-110° C.

N-Morpholinecarbonyl-L-phenylalanyl-L-homophenylalanine-α-(3-oxy-2-methyl-4-pyrone)methyl ketone (A12). MuPheHPheCH₂ Br (94 mg), potassium fluoride (45mg), and 3-hydroxy-2-methyl-4-pyrone was placed in a test tube underargon and 5 mL of dry DMF was added and the reaction was stirred at roomtemperature for two hours at which time the reaction showed a loss ofstarting material (silica gel, CHCl₃ /isopropanol, 9/1). The reactionwas then passed through a short plug of silica gel and the solvents wereremoved in vacuo. The product was then purified by size exclusionchromatography to give after evaporation of solvent a gold solid, mp.71-81.

N-Morpholinecarbonyl-L-phenylalanyl-L-homophenylalanine-α-(2-oxy-benzothiazole)methyl ketone (A13). MuPheHPheCH₂ Br (100 mg), 2-hydroxybenzothiazole(117 mg), and potassium fluoride (45 mg) were placed in a test tubeunder argon and 3 mL of dry DMF was added. The reaction was stirred atroom temperature until TLC (silica gel) showed loss of startingmaterial. The reaction was passed through a short silica gel column, thesolvents were removed in vacuo. The residue is dissolved in hot methanoland a white precipitate forms which upon filtration proves to be theproduct, mp. 211°-213° C.

N-Morpholinecarbonyl-L-phenylalanyl-L-homophenylalanine-α-(2-oxy-thiophene)methyl ketone (A14). MuPheHPheCH₂ Br (265 mg), potassium carbonate (119mg), and potassium fluoride (284 mg) were added together under argon andthen one gram of thiophenone in 4 mL DMF was added and the reaction wasallowed to stir at room temperature for 2 hours. The solvents wereremoved in vacuo and the products were separated on a 10 g silica gelcolumn.

N-Morpholinecarbonyl-L-phenylalanyl-L-homophenylalanine-α-(5-oxy-3-methyl-4-isoxazolecarboxylate)methyl ketone (A15). MuPheHPheCH₂ Br (510 mg), ethyl5-hydroxy-3-methyl-4-isoxazole carboxylate sodium salt, and 5 mL of DMFwas allowed to stir under argon at room temperature for 4 hours. Thereaction was then passed through a short plug of silica gel (CHCl₃/isopropanol) and the solvents were removed in vacuo. The product waspurified by size exclusion chromatography (LH-20) to give afterprecipitation in ether and filtration a white solid that melted with aphase change at 98°-105° and then again at 125°-131° C.

Formation of alkyl halide salts from inhibitors containing nitrogen inthe heterocycle leaving group: The methyl iodide isoquinoline salt ofN-Morpholinecarbonyl-L-phenylalanyl-L-homophenylalanine-α-(1-oxy-isoquinoline)methyl ketone (A16). Compound A8 (83 mg) is dissolved in 2 mL of tolueneand one mL of iodomethane is added. The reaction is sealed and allowedto stir for two days. A white precipitate forms which is filtered anddried under vacuum to give a white solid, mp. 159°-161° C.

    ______________________________________                                        CONSTRUCTION OF INHIBITORS WITH                                               OTHER HETEROCYCLES                                                            (IC.sub.50 Cathepsin B Inhibition)                                            ______________________________________                                        A1                                                                                  ##STR34##               (50 nM)                                         A2                                                                                  ##STR35##               (229 nM)                                        A3                                                                                  ##STR36##               (219 nM)                                        A4                                                                                  ##STR37##               (282 nM)                                        A5                                                                                  ##STR38##               (1,800 nM)                                      A6                                                                                  ##STR39##               (3800 nM)                                       A7                                                                                  ##STR40##               (569 nM)                                        A8                                                                                  ##STR41##               (457 nM)                                        A9                                                                                  ##STR42##               (4,500 nM)                                      A10                                                                                 ##STR43##               (4,500 nM)                                      A11                                                                                 ##STR44##               (252 nM)                                        A12                                                                                 ##STR45##               (385 nM)                                        A13                                                                                 ##STR46##               (21000 nM)                                      A14                                                                                 ##STR47##               (79000 nM)                                      A15                                                                                 ##STR48##               (2190 nM)                                       A16                                                                                 ##STR49##               (5600 nM)                                       ______________________________________                                    

EXAMPLE 13

Synthesis of Inhibitors With Heterocycles in their Peptide Backbones

The reaction scheme shown below is a first method for the synthesis ofcysteine protease inhibitors with heterocycles in their peptidebackbones. The synthetic method is an adaptation from that of Amos B.Smith and Ralph Hirshman as disclosed in "Design and Synthesis ofPeptidomimetic Inhibitors of HIV-1 Protease and Renin," 37 J. Med. Chem.215. ##STR50##

The reaction schemes shown below illustrate a second method for thesynthesis of cysteine protease inhibitors with heterocycles in theirpeptide backbones. This synthetic method is an adaptation from that ofDamewood et al., "Nonpeptidic Inhibitors of Human Leukocyte Elastase,"J. Med. Chem, 3303. ##STR51##

    ______________________________________                                        B. Heterocycle at P.sub.1                                                     ______________________________________                                         ##STR52##                                                                     ##STR53##                                                                     ##STR54##                                                                     ##STR55##                                                                    (A cathepsin H inhibitor)                                                     ______________________________________                                         Reagents: (a) sodium ethoxide/ether; (b) cyanoacetamide, piperidine           acetate, water; (c) 48% HBr, acetic acid (d) diphenyl phosphoryl azide        (Aldrich), triethyl amine, then benzyl alcohol; (e) sodium hydride, DMF,      then tertbutyl bromoacetate, (f) trifluoroacetic acid in methylene            chloride; (g) Nmethyl morpholine, isobutyl chloroformate then an unblocke     amino acid alpha oxyheterocycle methyl ketone such as                         Lhomophenyl-alpha-4-oxy-dihdro- furan2-one) methyl ketone (example 9); (h     Nmethyl morpholine, isobutyl chloroformate, then diazomethane/ether from      Diazald (Aldrich); 30% HBr/acetic acid in methylene chloride; (j)             potassium fluoride, DMF, oxyheterocycle such as tetronic acid; (k)            hydrochloric acid in dioxane.                                                 Note that this one amine acid fragment can be condensed as above with         another blocked amino acid fragment (BP.sub.2) to produce a cathepsin B,      type inhibitor.                                                          

EXAMPLE 14

Protocol for Testing ICE Inhibitors

The percent inhibition of two inhibitors,N-Benzoxycarbonyl-Valyl-Alanyl-Aspartyl-α-(4-oxy-(6-methyl-2-pyrone)methyl ketone, and N-Morpholinecarbonyl-L-Valyl-L-Alanyl-Aspartyl(OtBu)-α-(ascorbityl) methyl ketone, on IL-1β protease was determined asfollows. A 10 mM dithiothreitol, 100 mM Hepes, 10% sucrose, 0.1% CHAPS,pH 7.5 buffer solution with 50 μM Z-YVAD-AFC substrate was prepared. Theenzyme was activated for 1 minute in the buffer/substrate solution atroom temperature. Inhibitor was prepared as stock solution in dimethylsulfoxide. Inhibitor and enzyme/buffer were incubated for 15 minutes at37C. Final concentrations of inhibitor were 2000 nM, 200 nM, and 20 nM.Enzyme activity was followed by the release of free fluorescentdetecting group over sixty minutes at 37C, as compared to the control.

EXAMPLE 15

Protocol for Testing Calpain Inhibitors

The percent inhibition of one inhibitor,Acetyl-Leucyl-Leucyl-Phenylalanyl-α-(4-oxy-dihydrofuran-2-one) methylketone, on calpain (Calcium Activated Neutral Protease) was determinedas follows. A 50 mM Hepes, 10 mM calcium chloride, 5 mM cysteine, 1 mMβ-mercaptoethanol, pH 7.5 buffer solution was prepared. The enzyme wasactivated for 1 minute in the buffer solution at room temperature.Inhibitor was prepared as stock solution in dimethylformamide. Inhibitorand enzyme/buffer were incubated for 30 minutes at 37° C. Finalconcentrations of inhibitor were 20 μM, 2 μM, and 200 μM. Enzymeactivity was followed with 200 μM Boc-Valnyl-Leucyl-Lysine-AFC substrateby the release of free fluorescent detecting group over minutes at 37°C., as compared to the control. The inhibitor showed activity againstthe enzyme at less than 2 μM.

While the invention has been illustrated and described in detail in thedrawing and foregoing description, the same is to be considered asillustrative and not restrictive in character, it being understood thatonly the preferred embodiment has been shown and described and that allchanges and modifications that come within the spirit of the inventionare desired to be protected.

What is claimed is:
 1. Cysteine protease inhibitors of the formula##STR56## where B is H or an N-terminal blocking group;each P_(n) is anamino acid residue not containing a heterocycle; R₁ is the amino acidside chain of the P₁ amino acid residue; m is 0 or a positive integer;R₁₀ is H or an optionally substituted alkyl, aryl, heteroaryl; and X isO.
 2. Cysteine protease inhibitors of the formula: ##STR57## where B isH or an N-terminal blocking group;each P_(n) is an amino acid residuenot containing a heterocycle; R₁ is the amino acid side chain of the P₁amino acid residue; m is 0 or a positive integer; R₅ and R₆ areindependently hydrogen, alkyl or acyl; and X is O.
 3. The cysteineprotease inhibitors of claim 2 wherein R₅ and R₆ are each hydrogen.